OsSCL30 overexpression reduces the tolerance of rice seedlings to low temperature, drought and salt

Rice is one of the main food crops for the world population. Various abiotic stresses, such as low temperature, drought, and high salinity, affect rice during the entire growth period, determining its yield and quality, and even leading to plant death. In this study, by constructing overexpression vectors D-163 + 1300:OsSCL30 and D-163 + 1300-AcGFP:OsSCL30-GFP, the mechanism of action of OsSCL30 in various abiotic stresses was explored. Bioinformatics analysis showed that OsSCL30 was located on the chromosome 12 of rice Nipponbare, belonging to the plant-specific SCL subfamily of the SR protein family. The 1500 bp section upstream of the open reading frame start site contains stress-related cis-acting elements such as ABRE, MYC, and MYB. Under normal conditions, the expression of OsSCL30 was higher in leaves and leaf sheaths. The results of reverse transcription polymerase chain reaction showed that the expression of OsSCL30 decreased after low temperature, drought and salt treatment. In root cells OsSCL30 was localized in the nuclei. The results of the rice seedling tolerance and recovery tests showed that overexpression of OsSCL30 diminished the resistance to low temperature, drought and salt stresses in transgenic rice and resulted in larger accumulation of reactive oxygen species. This study is of great significance for exploring the response mechanisms of SR proteins under abiotic stresses.


Results
Bioinformatics analysis of OsSCL30. The OsSCL30 gene is located on rice chromosome 12, with an open reading frame of 792 bp, encoding 263 amino acids. It belongs to the plant-specific SCL subfamily of the SR protein family. In order to understand the relationship of OsSCL30 sequences among different species, the species with high amino acid sequence similarity were selected for cluster analysis. The OsSCL30 was found to be clustered with panicgrass (Panicum hallii), broomcorn millet (Panicum miliaceum), millet (Setaria italica), and maize (Zea mays), with a strong genetic relationship (Fig. 1a). Using the PlantCARE online website to analyze about 1500 bp upstream of ATG, it was found that the promoter of the OsSCL30 gene contains various elements related to stress response (Fig. 1b). These elements include ABRE (ABA responsive element), G-box (lightresponsive element), TGA-element (auxin-responsive element), GCN4_motif (involved in endosperm expression), TC-rich repeats (involved in defense and stress response), MYB (drought-related response element), etc.
Relative expression of OsSCL30. In order to select overexpressed lines for subsequent experiments, the expression level of OsSCL30 was detected by qRT-PCR. The results (Fig. 2g) showed that compared with WT, the www.nature.com/scientificreports/ expression levels of OE-1 and OE-3 were up-regulated the most, which were 197 times and 155 times, respectively. Therefore, OE-1 and OE-3 were selected as the subsequent experimental lines.
To detect the tissue-specific expression pattern of OsSCL30 gene, different untreated tissues (root, stem, leaf, sheath and spikelet) were collected, and the expression level of OsSCL30 in different tissues was detected by qRT-PCR. The results (Fig. 2h) showed the expression of OsSCL30 was highest in leaves, followed by leaf sheaths.
OsSCL30 is activated by abiotic stress. Because a variety of stress response factors were found in the promoter of OsSCL30, different stresses were applied to rice seedlings, and the expression level of OsSCL30 was detected by qRT-PCR. The results showed that the expression level of OsSCL30 in the control group was similar to the initial value during the whole process (Fig. 2a). The expression of OsSCL30 increased briefly after the low temperature treatment was imposed, rising to the highest level at 1 h, and then gradually decreased (Fig. 2b). The change in the whole period was not significant. The expression of OsSCL30 began to decrease immediately after imposition of the high temperature treatment, decreasing to a minimum at 2 h, and then gradually increased, but was lower than the initial value throughout the treatment (Fig. 2c). Under salt stress, the expression of OsSCL30 decreased significantly, then increased with the treatment duration, reaching the maximum at 4 h and then began to decrease, but the expression was lower than the initial value during the whole treatment duration (Fig. 2d). Under exogenous ABA treatment, the expression level of OsSCL30 was similar to that under salt treatment (Fig. 2e). After 0.5 h of drought treatment, the expression of OsSCL30 decreased briefly, then increased gradually, reaching a peak at 4 h, and then decreased sharply with prolonged treatment duration (Fig. 2f). In summary, various abiotic stresses can reduce the expression of OsSCL30 gene; among them, drought stress has the greatest impact on the OsSCL30 expression.
Subcellular localization of OsSCL30. The WOLF PSORT online website predicts that the OsSCL30 protein is localized in the nucleus. To further examine the subcellular localization of OsSCL30 protein in rice, the roots of transgenic rice plants OsSCL30-GFP were stained with DAPI and observed by confocal microscopy. The results showed that the GFP signal of the SCL30-GFP fusion protein was visible only in the nucleus, consistent with the prediction (Fig. 1c). Hence, we concluded OsSCL30 protein was localized in the nucleus of rice.
Overexpression of OsSCL30 attenuates low temperature resistance in transgenic rice. To verify the response of OsSCL30 overexpression to low temperature stress, we cultured 5-day-old Nip (WT) and two overexpression lines (OE-1, OE-3) at 4 °C for 3 days followed by 7-day recovery. The results showed that the growth of OsSCL30-OE was similar to that of WT seedlings under normal growth conditions, but low temperature inhibited the growth of OsSCL30-OE seedlings (Fig. 3a). The plant height of OsSCL30-OE seedlings after low temperature treatment was significantly shorter than that of WT, but the root length was significantly longer than that of WT (Fig. 3b).
We cultured 14-day-old Nip (WT) and two overexpressing lines (OE-1, OE-3) at 4 °C for 7 days and then allowed them to recover for 12 days. The wilting of OsSCL30-OE seedlings after low temperature treatment was more severe than that of WT (Fig. 3d). After the recovery period, the survival rate of OsSCL30-OE seedlings (24-25% for the two lines) was significantly lower than that of WT (85%) (Fig. 3e-f).
In order to clarify the regulatory pathway of OsSCL30, we detected the expression of important genes in the cold response pathway of OsSCL30 overexpressed plants. The results showed that the core components of rice ICE-CBF pathway 29 , including OsCBF2 and OsCBF3, were down regulated in OsSCL30-OE compared with WT ( Fig. 3g-h). In conclusion, overexpression of OsSCL30 reduced the low temperature resistance of transgenic rice plants.
Overexpression of OsSCL30 attenuates drought resistance in transgenic rice. To verify the response of OsSCL30 overexpression to drought stress, we cultured 5-day-old Nip (WT) and two overexpressing lines (OE-1, OE-3) in 15% w/v PEG for 3 days followed by 7-day recovery. The OsSCL30-OE grew similarly to WT seedlings under normal growth conditions, but drought inhibited the growth of OsSCL30-OE seedlings (Fig. 4a). After drought treatment, the plant height and root length of transgenic lines were shorter (root length was significant in OE-1 and not significant in OE-3) compared with WT (Fig. 4b).
To exacerbate the drought stress, we cultured 14-day-old Nip (WT) and two overexpressing lines (OE-1, OE-3) in 18% w/v PEG environment for 8 days and then allowed them to recover for 12 days. The results showed that the severity of wilting was similar in OsSCL30-OE and WT seedlings after 8 days of 18% w/v PEG treatment (Fig. 4d). However, after the recovery period, the survival rate of OsSCL30-OE seedlings (32-36% for the two OE lines) was significantly lower than that of WT (98%) (Fig. 4e-f).
In order to explore the regulatory mechanism of OsSCL30 under drought stress, we detected the expression of important genes in the drought response pathway of OsSCL30 overexpressing plants. The results showed that the expression of drought related response genes OsDREB2A 30 and OsNAC6 31 in rice OsSCL30-OE was down regulated compared with WT ( Fig. 4g-h).
In addition, compared with WT, the water loss rate of detached leaves of OsSCL30-OE was significantly higher, indicating that the water holding capacity of OsSCL30-OE was weaker (Fig. 4i). In conclusion, overexpression of OsSCL30 reduced the drought resistance of transgenic rice plants.
Overexpression of OsSCL30 weakens salt resistance in transgenic rice. To verify the response of OsSCL30 overexpression to high salt stress, we cultured 5-day-old Nip (WT) and two overexpressing lines (OE-1, OE-3) in 120 mM NaCl for 3 days followed by recovery for 7 days. The OsSCL30-OE grew similarly to www.nature.com/scientificreports/ WT seedlings under normal growth conditions, but high salt inhibited the growth of OsSCL30-OE seedlings (Fig. 5a). After salt treatment, both plant height and root length of OsSCL30-OE seedlings were significantly shorter than those of WT (Fig. 5b). We cultured 14-day-old Nip (WT) and two overexpressing lines (OE-1, OE-3) in 150 mM NaCl for 6 days and then allowed them to recover for 12 days. The results showed that wilting after 6 days of 150 mM NaCl treatment were analyzed after two days of treatment at 4 °C, respectively. Error bars represent ± SE (n = 3). Asterisks indicate significant differences between transgenic lines and WT (*P < 0.05, **P < 0.01). www.nature.com/scientificreports/ was more severe in OsSCL30-OE than WT seedlings (Fig. 5d). After recovery, the survival rate of OsSCL30-OE seedlings (4-14% for the two lines) was significantly lower than that of WT (96%) (Fig. 5e-f).
In order to explore the regulatory mechanism of OsSCL30 under salt stress, we detected the expression of important genes in the salt response pathway of OsSCL30 overexpressing plants. The results showed that the expression of salt related response genes OsDREB2A 30 and OsNAC6 31 in rice OsSCL30-OE was down regulated (g-h) The expression levels of OsDREB2A and OsNAC6 were analyzed after two days of treatment at 150 mM NaCl, respectively. Error bars represent ± SE (n = 3). Asterisks indicate significant differences between transgenic lines and WT (*P < 0.05, **P < 0.01). www.nature.com/scientificreports/ compared with WT ( Fig. 5g-h). Therefore, we conclude the overexpression of OsSCL30 reduced the salt tolerance of transgenic rice plants.

Overexpression of OsSCL30 affects the accumulation and scavenging of ROS under different stresses.
To examine the effect of OsSCL30 overexpression on the accumulation of reactive oxygen species (ROS), we subjected 14-day-old Nip (WT) and two overexpressing lines (OE-1, OE-3) to different stresses, and performed NBT staining to estimate the accumulation of superoxide ions (O 2− ). In the control (no stress) treatment, the OsSCL30-OE leaves showed no significant difference from WT; by contrast, after the low temperature, drought and salt treatments the OsSCL30-OE leaves had numerous black spots (indicating more ROS accumulation and more severe oxidative damage) compared with WT (Fig. 6a). As an indication of cellular oxidative damage, the content of MDA showed no significant difference between OsSCL30-OE and WT in the control treatment without stress, whereas after low temperature, drought and salt treatments OsSCL30-OE accumulated more MDA and suffered more severe oxidative damage than WT (Fig. 6b).
The activities of reactive oxygen species scavenging enzymes (SOD, CAT and POD) were similar between OsSCL30-OE and WT without any stress imposed, whereas these activities were significantly lower in Figure 6. (a) Nitroblue tetrazolium (NBT) staining was used to detect the levels of superoxide anion in wild type (WT) and OE lines before and after low temperature (4 °C for 2 days), drought (20% w/v PEG for 2 days), and salt (150 mM NaCl for 2 days) treatments. (b-e) Detection of reactive oxygen species scavenging enzyme activities in wild type (WT) and OE lines before and after low temperature (4 °C for 2 days), drought (20% w/v PEG for 2 days) and salt (150 mM NaCl for 2 days). Error bars represent ± SE (n = 3). Asterisks indicate significant differences between transgenic lines and WT (*P < 0.05, **P < 0.01). www.nature.com/scientificreports/ OsSCL30-OE than WT after the low temperature, drought and salt treatments, indicating relatively poor capacity of the OsSCL30-OE lines to scavenge reactive oxygen species (Fig. 6c-e).
In addition, we also analyzed the changes in expression levels of genes related to reactive oxygen species production and scavenging under cold, drought, salt stress and normal conditions, including OsRbohA (NADPH oxidase), OsCu-ZnSOD2 (superoxide dismutase), OsPOD (peroxidase) and OsCATA (catalase) 32,33 . Under normal conditions, there was no significant difference in the expression of OsRbohA, OsCu-ZnSOD2, OsPOD and OsCATA between WT and OsSCL30-OE. However, under stress treatment, compared with WT, the expression of OsRbohA in OsSCL30-OE increased in varying degrees (Fig. 7a-c), and the expression of OsCu-ZnSOD2, OsPOD and OsCATA decreased in varying degrees (Fig. 7d-l). In conclusion, under low temperature, drought and salt stresses, overexpression of OsSCL30 can reduce the activity of enzymes scavenging reactive oxygen species, resulting in aggravated oxidative damage.
Relative expression of OsSCL30 homologous proteins. It is an established fact that members of SR protein gene family play a crucial role in regulating proteome diversity in spatio-temporal manner. Since the SR protein family consists of multiple members, we studied the effect of OsSCL30 overexpression on other SCL subfamilies in transgenic lines (OsSCL30A, OsSCL25, OsSCL26, OsSCL28, OsSCL57) 34 . The results showed that the expression of its homologous proteins increased, and the increase of OsSCL30A was the most significant (Fig. 7m). The overexpression of OsSCL30 negatively regulated the abiotic stress tolerance of plants, which may be due to crosstalk with other members of the family, and the cumulative effect leaded to the sensitivity to abiotic stress.

Analysis of agronomic traits in transgenic rice overexpressing OsSCL30. Compared with WT,
OsSCL30-OE had obvious difference in grain size. Among them, grain length was significantly longer than WT, increasing by 13.5%, grain width was significantly smaller than WT, decreasing by 13.2%, grain thickness was significantly smaller than WT, decreasing by 4.3%, and 1000-grain weight was slightly larger than WT, but did not reach a significant level. In addition, the color of the glume of OsSCL30-OE was darker than that of WT, and the color of the endosperm was cloudier (Fig. 8). The results of field trait measurement showed that compared with WT, the plant height, tiller number, effective spikes and yield per plant of OsSCL30-OE were significantly reduced by 8.3%, 45.5%, 46.7% and 49.9%, respectively (Table 1).

Discussion
Abiotic stresses such as drought, salt and extreme temperatures have a huge impact on world agricultural production. Plants typically respond and adapt to these stresses at the levels ranging from molecular to cellular and organ levels, encompassing a range of physiological and biochemical processes [35][36][37][38] . Understanding the complexity of the mechanisms by which plants respond to abiotic stress is crucial for developing high-yielding crops. The results of this study showed that OsSCL30 overexpressing plants had reduced tolerance to low temperature, drought and salt.
The SR proteins are nucleophosmin proteins with characteristic Ser/Arg-rich domains and one or two RNA recognition motifs 39 .They are a very important class of splicing regulators and can be divided into six subfamilies according to their structural characteristics, namely: SR, RSZ, SC, SCL, RS2Z and RS subfamilies, of which SCL, RS2Z and RS are three subfamilies unique to plants 34,40 . The SR protein family in plants had been confirmed to have a role of alternative splicing in stress responses 41 . The expression of AtSR45a was induced by strong light exposure, and the expression of AtSR30 was increased by strong light exposure and plastoquinone (PQ) or salinity treatment, and decreased by low temperature 42 . Overexpression of Populus trichocarpa PtSCL30 in Arabidopsis reduced cold tolerance, possibly due to alternative splicing (AS) changes in genes critical for cold tolerance such as ICE2 and COR15A 43 . The cassava MeSCL30a overexpression lines were hypersensitive to salt and drought stress, and had lower germination and greening rates 27 . In the present study, the OsSCL30 (Os12g38430) (SCL subfamily) codes for the protein located in the nucleus and contains an N-terminal RNA recognition sequence. Low temperature, drought and salt stresses all decreased the expression of OsSCL30, with drought stress having the greatest effect on the expression of OsSCL30 (Fig. 2a-f), indicating that OsSCL30 may be involved in the abiotic stress responses. The overexpression of OsSCL30 reduced the survival rate of transgenic plants under low temperature, drought and salt stresses (Figs. 3, 4 and 5). Under stress, the expression of positive regulatory genes in stress-related response in OsSCL30-OE was down regulated compared with WT (Figs. 3, 4 and 5). The overexpression of OsSCL30 affected the expression of SR genes (OsSCL30A, OsSCL25, OsSCL26, OsSCL28 and OsSCL57) of other SCL subfamilies in the transgenic line, and the expression of OsSCL30A increased most significantly (Fig. 7m). These results suggest that OsSCL30 may participate in the splicing of mRNAs, produce proteins with changed function and structure, and negatively regulate the tolerance of plants to low temperature, drought and saline alkali. In addition, OsSCL30 may have crosstalk with other members of the family and produce cumulative effects, thus affect the tolerance of transgenic plants to abiotic stress.
Plants subjected to abiotic stress accumulate ROS 44 , that can cause oxidative damage to cell membranes, proteins, DNA molecules, etc [45][46][47] . The activities of antioxidant enzymes SOD, CAT and POD, as well as membrane lipid peroxidation (generating MDA) can reflect the degree of damage to plants caused by stress to a certain extent. SOD can scavenge superoxide anion free radicals, CAT and POD can catalyze the decomposition of H 2 O 2 into H 2 O and O 2 , which can help plants resist peroxidation and is positively related to the tolerance of various stresses [48][49][50] . As a product of ROS lipid peroxidation, MDA is often used to indicate a degree of cell membrane lipid peroxidation 51,52 . Overexpression of Cassava MeSR34 enhanced salt stress tolerance in transgenic Arabidopsis by maintaining ROS homeostasis and affecting the CBL-CIPK pathway 53  www.nature.com/scientificreports/ sensitivity of transgenic plants to exogenous ABA 54 . In the study presented here, the NBT staining results of OsSCL30-OE and WT before the three stress treatments were similar, and the NBT-stained leaves of OsSCL30-OE after stress treatment showed more black spots than WT (Fig. 6a), indicating that the OsSCL30-overexpressing lines accumulated more ROS. Furthermore, the activities of antioxidative enzymes under non-stress conditions were similar in the OsSCL30-overexpressing lines and WT, but under three stress conditions (low temperature, drought, high salt), the activities of SOD, POD and CAT were lower in the OsSCL30 overexpression lines than WT (Fig. 6c-e). The content of MDA was higher in the OsSCL30-overexpressing lines than WT (Fig. 6b). Under normal conditions, there was no significant difference in the expression of OsRbohA, OsCu-ZnSOD2, OsPOD and OsCATA in WT and OsSCL30-OE. However, under stress treatment, compared with WT, the expression of OsRbohA in OsSCL30-OE increased in varying degrees (Fig. 7a-c), and the expression of OsCu-ZnSOD2, OsPOD and OsCATA decreased in varying degrees (Fig. 7d-l). These findings suggested that overexpression of OsSCL30 may alter the splicing pattern of pre-mRNA to generate proteins with multiple functions and structures, so as  www.nature.com/scientificreports/ to regulate ROS scavenging activity, cause transgenic rice plants to suffer more severe membrane damage and reduce their tolerance to cold, drought and salt stress. The OsSCL30 was localized mainly in the nucleus, with tissue-specific expression (higher in leaves and leaf sheaths than roots, stems and spikelets). Under stress, OsSCL30-overexpression resulted in more severe membrane damage in transgenic rice plants by decreasing the reactive oxygen species scavenging activity, thereby reduced tolerance to cold, drought and salt stress. The results of field trait measurement showed that the effective spikes and yield per plant of OsSCL30-OE were significantly lower than those of WT, and the reduction ratio of yield per plant was the most obvious. Characterization of the negative role of OsSCL30 in rice stress provides a theoretical basis for breeding more stress-tolerant rice using CRISPR/Cas9 gene modification system in the future.

Materials and methods
Plant materials and growth conditions. In this study, the japonica rice Nipponbare (Oryza sativa ssp. japonica 'Nipponbare') was used as the experimental material. We have been granted permission to collect Oryza sativa ssp. japonica 'Nipponbare' . Transgenic rice plants were obtained by agrobacterium tumefaciens mediated genetic transformation 55 . The seeds were soaked in water for 1 day, then surface sterilized by soaking in 2% v/v NaClO for 30 min and rinsing with sterile water. Afterwards, seeds were spread on moist filter paper in a Petri dish to promote germination. When the shoots grew to 5 mm, the seedlings were transplanted into rice nutrient solution and grown at 28 °C/22 °C and 16 h light/8 h dark cycle 56 . To study the effect of abiotic stress conditions on the expression of OsSCL30, after 2 weeks of culture, uniformly sized seedlings were selected for stress treatments, including cold (4 °C), salt (150 mM NaCl) and drought (15% w/v PEG 6000). All the experiments were performed in accordance with relevant guidelines and regulations.
Total RNA extraction and cDNA synthesis. Fresh leaves were sampled from the normal and stress treatments, snap-frozen in liquid nitrogen, and used for total RNA extraction according to the operating instructions of the RNA extraction kit (BioFlux). RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa) and was stored at -20 °C.
Quantitative analysis of OsSCL30 under abiotic stress. Two-week-old rice seedlings were subjected to cold, drought and salt stress treatments, and leaf tissue was harvested at the indicated times for extraction of total RNA and reverse transcription. This cDNA was diluted and used as a template to analyze the gene expression of OsSCL30 under abiotic stress. Primers were designed for OsSCL30 gene and the rice reference gene UBQ (ubiqutin) using Primer Premier 5 software (UBQ-F:5′-AAC CAG CTG AGG CCC AAG A-3′; UBQ-R:5′-ACG ATT GAT TTA ACC AGT CCATG-3′; OsSCL30-qRT -F:5′-GTC TCG TTC CCG TTCTC-3′; OsSCL30-qRT -R:5′-GTA GTC ATC TCG CCG TCT -3′).
Analysis and cloning of the OsSCL30 gene. To obtain the OsSCL30-overexpression vector, the CDS sequence of OsSCL30 was obtained from the Rice Genome Database (rapdb). The primer for OsSCL30 gene was designed using software Primer Premier 5 (OsSCL30-F:5′-tggagaggacagcccaagcttATG AGG AGG TAC AGC CCA CCA -3′; OsSCL30-R:5′-gtaccgaattcccggggatccTCA GTC GCT GCG GGC AGG -3′). The amplified target fragments were purified and recovered with a gel recovery kit. The expression vector (D-163 + 1300) was double digested with Hind III and BamH I endonucleases, followed by recombination to construct an OsSCL30 overexpression vector.
Analysis and cloning of promoter. The sequence information about 1.5 kb upstream of the OsSCL30 open reading frame was obtained in NCBI (https:// www. ncbi. nlm. nih. gov/), and then passed through Plant-Care (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/) for analysis of cis-acting elements in the OsSCL30 promoter.
Genetic relationship analysis of OsSCL30 gene. In order to understand the phylogenetic relationships of OsSCL30 sequences, species with high amino acid sequence similarity were selected and clustered by software MEGA5.0. Subcellular localization of OsSCL30. Seeds of the OsSCL30-GFP transgenic plants were germinated in the dark, stained with nuclear dye (DAPI), and observed using confocal microscopy.

Transgenic plants treated with abiotic stress.
To study the effect of low temperature on overexpressing plants, we cultured 2-week-old plants at 4 °C for 7 days and then allowed them to recover at 28 °C for 12 days.
To study the effect of drought on overexpressing plants, we cultured 2-week-old plants at 18% w/v PEG 6000 for 8 days followed by recovery at 28 °C for 12 days. To study the effect of high salt on overexpressing plants, we cultured 2-week-old plants in 150 mM NaCl for 6 days and then allowed them to recovery at 28 °C for 12 days. For all stress treatments plants were grown in the rice nutrient solution.
NBT staining. Two-week-old seedlings were treated at 4 °C, 20% w/v PEG 6000, or 150 mM NaCl for www.nature.com/scientificreports/ Measurement of the physiological parameters. Physiological parameters were determined after 2-week-old seedlings were treated for 2 days at 4 °C, 20% w/v PEG 6000 or 150 mM NaCl. The MDA content was determined by spectrophotometry 58 . The activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were determined as described elsewhere 59 .
Water loss rate. The measurement of water loss rate was as described by predecessors 60 . The leaves of 14-day-old rice seedlings were sampled at room temperature. The leaves were placed on filter paper on the experimental bench and weighed at specified time. Then calculate the percentage of water loss. Three biological repeats were performed on each line.

Statistical analysis.
All experiments were repeated three times, and the results were consistent. The data were processed and analyzed by the t-test, and the difference was statistically significant at P < 0.05(*) or P < 0.01(**).

Data availability
All data generated or analysed during this study are included in this published article.